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Gene Expr Patterns
2003 Aug 01;34:521-4. doi: 10.1016/s1567-133x(03)00055-3.
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Xenopus Germes encodes a novel germ plasm-associated transcript.
Berekelya LA
,
Ponomarev MB
,
Luchinskaya NN
,
Belyavsky AV
.
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A cDNA tag specific for the vegetal pole of early Xenopus embryo was used to isolate a novel cDNA using RACE technique. The corresponding mRNA demonstrated a localization pattern typical for the germ plasm-associated messages, and was, therefore, named Germes. The open reading frame of Germes encodes a predicted 68 kDa protein with two leucine zipper motifs and a putative EF-hand domain, but otherwise no substantial homology to known proteins. In situ hybridization analysis shows that Germes transcript localizes to the vegetal cortex via the mitochondrial cloud early in oogenesis and segregates with the germ plasm during early embryogenesis. Our data indicate that Germes is a novel germ plasm-specific RNA.
Fig. 3. Localization of Germes mRNA during oogenesis analyzed by in situ hybridization. (A) Whole-mount of stages I–VI oocytes, vegetal view. In the smallest oocytes (stage II), the entire cytoplasm is heavily stained. (B) Epon section through stage II oocytes reveals concentration of Germes message in mitochondrial cloud. Oocyte on the right illustrates the commencement of the accumulation of the message in the vegetal cortex. (C) In situ hybridization for Germes on paraffin sections of stage III oocyte. The mRNA anchors on the vegetal cortex, forming the continuous layer. (D) In situ hybridization on paraffin sections of stage VI oocytes. The Germes transcript forms a granular pattern within the vegetal cortex. mc, mitochondrial cloud; n, nucleus; vc, vegetal cortex.
Fig. 4. Analysis of localization of Germes mRNA during early Xenopus development by in situ hybridization. (A,B) Whole-mount hybridization of two- (A) and eight-cell (B) stage embryo, vegetal view. Germes RNA localizes within small individual granules at the vegetal pole of two-cell stage embryo (A). These granules appear to aggregate into lager complexes in the vegetal cortex of eight-cell embryo (B). (C) Transparent Epon block of early blastula, containing half of the embryo and viewed from above. Small Germes RNA-staining islands have coalesced into a few aggregates, settling inside the vegetal cells. (D,E) Epon sections of eight-cell (D) and early blastula (E) (same embryo as in (C)) stages. Aggregating granules have lost their connection with the vegetal cortex and change their position inside the cells. (F) Epon section of mid-gastrula stage. There is a Germes RNA-positive PGC in endoderm mass. Higher magnification of the same slide (G) shows perinuclear localization of the transcript. Compare yolk-free stained perinuclear region of PGC (G) with yolk-reach cytoplasm of endodermal cells (H). bcl, blastocoel; gcl, gastrocoel; nuclei are marked by arrows. The border of PGC, discernible at higher resolution, is marked by arrowheads.
Fig. 2. Molecular analysis of Germes expression. (A) Northern blot analysis of Germes RNA expression during embryogenesis. (B) Northern hybridization of Germes with RNA from adult tissues. The stages or tissues tested are indicated at the top of each line. Membranes were first hybridized with the Germes probe, then stripped and reprobed with EF-1α probe as a loading control.
